Use of interleukin-2 (IL-2) receptor-specific agents to treat allograft rejection

ABSTRACT

A method of lysing unwanted, non-malignant cells in a mammal, the cells having on their surfaces a receptor for a growth factor, and the method including administering to the mammal a cell-lysing amount of a substance characterized in that it has specific affinity for the receptor of the growth factor and has the ability to effect the lysis of the cells.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided by the federalgovernment, which has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/469,538filed Jun. 6, 1995, now U.S. Pat. No. 5,607,675, which is a continuationof application Ser. No. 08/275,010 filed Jul. 8, 1994, now U.S. Pat. No.5,510,105, which is a divisional of application Ser. No. 07/842,463filed Feb. 27, 1992, now U.S. Pat. No. 5,336,489, which is acontinuation of application Ser. No. 07/692,830, filed Apr. 26, 1991,now abandoned, which is a continuation of application Ser. No.07/492,616 filed Mar. 12, 1990, now abandoned, which is a continuationof application Ser. No. 06/772,893, filed Sep. 5, 1985, now U.S. Pat.No. 5,011,684.

BACKGROUND OF THE INVENTION

This invention relates to the lysis of unwanted, non-malignant cells,e.g., lymphocytes involved in the rejection of allografts such astransplanted organs.

Allograft rejection is an immune response, involving activatedT-lymphocytes. Currently used immunosuppressive protocols designed toinhibit rejection involve the administration of drugs such asazathioprine, cyclosporine, and corticosteroids, all of which causetoxic side-effects to non-lymphoid tissues. The recent development ofpan-T-lymphocyte monoclonal antibodies represents an importantrefinement in therapy, since only T-lymphocytes are targeted by theadministration of such antibodies. However, this therapy has thedisadvantage of destroying, along with the T-lymphocytes involved inallograft rejection, those required for normal immune surveillance.

SUMMARY OF THE INVENTION

In general, the invention features a method of lysing unwanted,non-malignant cells in a mammal, which cells have on their surfaces areceptor for a growth factor; the method substance characterized in thatit has specific affinity for the growth factor receptor and has theability to effect lysis of the cells. (As used herein, "malignant" cellsrefers to cancerous cells, e.g., primary or metastatic solid tumorcells, or leukemia cells; the non-malignant cells targeted according tothe invention are unwanted cells which are not cancerous. "Growthfactor" refers to a substance which, when taken into a cell afterbinding to a growth factor receptor on the surface of the cell,facilitates proliferation of the cell. "Specific affinity" refers to theability of a substance to bind virtually exclusively to a particulargrowth factor receptor, e.g., the interleukin-2 ("IL-2") receptor, andnot to other cell surface receptor proteins, e.g., insulin receptors.)

In preferred embodiments, the unwanted cells are lymphocytes, i.e.,T-lymphocytes or B-lymphocytes, and the growth factor receptor-specificsubstance includes either an antibody (preferably a monoclonal antibodyof the lytic IgG or IgM isotypes) or the growth factor itself (or areceptor-specific analog thereof) linked to a cytotoxin, e.g.,diphtheria toxin or ricin, via either a convalent linkage such as adisulfide linkage or, more preferably, via a peptide linkage.

The unwanted, non-malignant cells most preferred to be lysed by themethod of the invention are T-lymphocytes, which are the cell typeprimarily responsible for causing rejection of allografts (e.g.,transplanted organs such as the heart). T-lymphocytes (killer andhelper) respond to allografts by undergoing a proliferative burstcharacterized by the transitory presence on the T-lymphocyte surfaces ofIL-2 receptors. Killing these cells by the administration, during theproliferative burst, of a lytic, IL-2 receptor-specific substanceinhibits allograft rejection, and also advantageously fails to adverselyaffect other cells (including resting or long-term memory T-lymphocytesneeded for fighting infections), since these other cells do not bearIL-2 receptors and are therefore not recognized by the IL-2receptor-specific substance. In addition, cell lysis according to theinvention is efficient because the IL-2 receptor binds to IL-2receptor-specific substances so that the cytotoxin, if one is involved,is internalized in a way which results in cell death.

Where the lytic substance is an antibody of the complement-fixing IgG orIgM isotypes, it is not necessary that the antibody compete with IL-2for the IL-2 receptor; i.e., the antibody can be one which binds to thereceptor in a way which permits IL-2 to bind as well. Competitivebinding is, however, important, for non-lytic IL-2 receptor-specificsubstances, as is discussed below.

The concept that allograft rejection can be inhibited by takingadvantage of the proliferative burst of attacking T-lymphocytes which ischaracterized by the transient presence of IL-2 receptors on thesurfaces of the T-lymphocytes can also form the basis for inhibitingallograft rejection using an IL-2 receptor-specific substance which, byvirtue of its binding to the IL-2 receptors of the T-lymphocytes whichwould otherwise attack the allograft, impairs their ability to causerejection of the allograft, but does not lyse them via complementfixation. Such substances, (e.g., non-complement fixing IgA antibodies),to be effective, must compete with IL-2 for the IL-2 receptor, so thattheir administration will prevent IL-2 from binding to theT-lymphocytes.

This preventing of the binding of IL-2 to T-lymphocytes can result inseveral important phenomena which contribute to rejection inhibition.First, the T-lymphocytes, newly activated by the presence of theallograft, fail to proliferate, and eventually die, due to the lack ofthe essential anabolic stimulus IL-2. In addition, the T-lymphocytes,deprived of IL-2, fail to release at least three lymphokines which playimportant roles in organ rejection. One of these, gamma interferon,normally activates allograft-attacking macrophages, and also stimulatesthe allograft to produce additional antigen, marking the allograft formore vigorous attack by the immune system. A second of these lymphokinesis B-cell differentiation factor II, which ordinarily would stimulatethe differentiation of B-cells, which in turn would produceallograft-attacking antibodies. A third lymphokine is IL-3, ahematopoietic stem cell growth factor also believed to play an importantrole in allograft rejection.

The method of the invention inhibits allograft rejection in a mannerwhich does not cause general immune suppression, with its resulting riskof life-threatening infections. In addition, the method sparesdonor-specific T suppressor cells, which can thus proliferate and aid inprolonging allograft survival. Furthermore, antibodies do not need to betailored to individual patients; a single antibody can be used as auniversal allograft rejection inhibiting agent for every donor-recipientcombination. In addition, therapy need not be continuous following theallograft, but can be discontinued after a course of treatment.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawing will first be described.

DRAWING

In the drawing, the FIGURE is a graph showing skin allograft survivalfollowing treatment with an antibody according to the invention (opencircles with dashed line, n=9) and untreated controls (closed circleswith solid line, n=7).

ANTIBODY THRAPY

One embodiment of the invention employs, as the IL-2 receptor-specificsubstance, an antibody (preferably monoclonal) which is specific for theIL-2 receptor on T-lymphocytes and is which is preferably capable ofeffecting in vivo lysis of T-lymphocytes to whose IL-2 receptors itbinds. Antibodies specific for the IL-2 receptor on T-lymphocytes can bemade using standard techniques, or can be purchased, e.g., from BectonDickenson Company (mouse-human monoclonal anti-IL-2 receptor antibodies,packaged in azide, which would need to be removed prior to use). Theantibody can be used alone, or it can be coupled to a toxin to increaseits lytic capacity. The antibody can be monoclonal or polyclonal, andcan be derived from any suitable animals. Where the antibody ismonoclonal and the mammal being treated is human, mouse-human anti-IL-2receptor antibodies are preferred.

Production and initial screening of monoclonal antibodies to yield thosespecific for the IL-2 receptor can be carried out as described inUchiyama et al. (1981) J. Immunol. 126 (4), 1393; this method, briefly,is as follows. Human cultured T-lymphocytes are injected into mammals,e.g., mice, and the spleens of the immunized animals are removed and thespleen cells separated and then fused with immortal cells, e.g., mouseor human myeloma cells, to form hybridomas.

The antibody-containing supernatants from the cultured supernatants arethen screened for those specific for the IL-2 receptor, using acomplement-dependent cytotoxicity test, as follows. Human T-lymphocytesand EBV transformed B-lymphocytes are labeled with ⁵¹ Cr sodium chromateand used as target cells; these cells are incubated with hybridomaculture supernatants and with complement, and then the supernatants arecollected and counted with a gamma counter. Those supernatantsexhibiting toxicity against activated T-lymphocytes, but not resting T-or B-lymphocytes, are selected, and then subjected to a furtherscreening step to select those supernatants containing antibody whichprecipitates (i.e., is specifically reactive with) the 50 kilodaltonglycoprotein IL-2 receptor (described in detail in Leonard et al. (1983)P.N.A.S. USA 80, 6957). The desired anti-IL-2 receptor antibody ispurified from the supernatants using conventional methods.

Graft rejection inhibition employing anti-IL-2 receptor monoclonalantibodies has been carried out using two different mammalian species,mice and rats, as described below.

MICE Animals and Operative Techniques

Inbred male mice weighing 20-25 grams of strains C57B1/10, B10.BR, andB10.AKM (Jackson Laboratory, Bar Harbor, Me.) were used throughout.These strains are completely mismatched for the H-2 locus, but share thesame genetic background.

Vascularized, heterotopic heart allografts were performed as originallydescribed by Corry et al. Transplantation (1973) 16, 343. The aorta wasanastomosed to the abdominal aorta, and the pulmonary artery to theadjacent vena cava using standard microvascular techniques with 10-0nylon suture (Ethicon, Inc., Somerville, N.J.) under 20×magnification.With completion of the anastomoses and warming of the heart withRinger's lactate solution at 37° C., normal sinus rhythm was resumed.Function of the transplant heart was assessed by daily palpation ofventricular contractions through the abdominal wall. Rejection wasdefined as the cessation of all mycardial contractions, which wasconfirmed at laparotomy under ether anesthesia.

To perform skin grafts, full thickness tail skin was removed from adonor sacrificed by cervical dislocation. The recipient was anesthetizedwith ether, shaven, and a graft bed prepared on the posterior flank byremoving a 0.5×0.5 cm portion of skin, with care taken not to injure theunderlying panniculus or its blood vessels. The graft was shaped to fitthe graft bed, positioned, and covered with baseline gauze. A bandagewas then wrapped circumferentially around the chest to protect thegraft. The grafts were inspected daily beginning on day 6 or 7, andconsidered rejected when greater than 50% of the epithelium wasnon-viable.

Preparation and Administration of Monoclonal Antibody

The monoclonal antibody employed was antibody M7/20, which is describedin Gaulton et al. (1985) Clin. Immunol. and Immunopath. M7/20 is amonoclonal rat anti-mouse κ, u, Ig antibody specific for the IL-2receptor. M7/20 was purified from the culture supernatants of cellsgrown in serum free media (Hanna Labs, Berkeley, Calif.). Supernatantswere precipitated with 40-50% saturated ammonium sulfate, dialyzed,passed over DEAE Affi-Gel Blue (Bio-Rad, Richmond, Va.) in 20 mM NaCl,and the eluate fractionated on Sephadex G-200 (Pharmacia, Piscataway,N.J.), run in 20 mM Tris (pH 7.2), 250 mM NaCl, 0.5% n-butanol. Antibodypurity was assessed by SDS-Page gel electropheresis. There was alsoemployed a control monoclonal antibody, RA3.2C2, not specific for theIL-2 receptor. The hybridoma producing RA3-2C2, which binds to pre-Bcells and to some mature B cells, was obtained from the American TypeCulture Collection (Rockville, Md.), and the antibody purified by theprocedure described above for M7/20.

Both antibodies were diluted to a final concentration of 25 μg/ml inphosphate buffered saline. Treated recipients of heart or skinallografts received 0.2 ml (5 μg) by intraperitoneal injection daily for10 days, usually beginning the day of transplant. In a small number ofheart graft recipients, the onset of treatment was delayed until day 3or 6, then given for a total of 10 daily doses.

Histology

Separate groups of treated and untreated C57B1/10 recipients of B10.BRheart allografts were sacrificed at intervals post-transplant forhistologic studies. Hearts were removed from two animals in each groupat days 3, 6, and 9 following trnasplantation, fixed in formalin,sectioned, and stained with hematoxylin and eosin.

Results

The results, given in Table I, below, demonstrate the ability of M7/20to prevent rejection of vascularized heart allografts in two straincombinations of inbred mice. Control C57B1/10 heart allografts inuntreated B10.AKM recipients were rejected with a median survival of 8days. However, when treatment with M7/20 was begun on the day oftransplant and continued for 10 days at a dose of 5 μg daily, 4 of 6grafts survived indefinitely (>90 days), with two rejecting at 20 and 31days. This survival is significantly longer than control (p<0.01).Similar results were obtained in C57B1/10 recipients of B10.BR heartallografts. Control grafts were rejected at 10-20 days, while treatedgrafts were not rejected until 20, 27, 34, 38 days, with two graftsfunctioning for more than sixty days (p<0.01). Treatment with RA3-2C2did not prolong graft survival.

                  TABLE I    ______________________________________    The effect of M7/20 on survival of murine heart    allografts.    Recipient           Donor     Treatment  Allograft Survival in Days    ______________________________________    B10.AKM           C57B1/10  none       8,8,8,8,16,29    B10.AKM           C57B1/10  M7/20.sup.a                                20,31,>90,>90,>90,>90    B10.AKM           C57B1/10  RA3-2C2.sup.a                                6,9,9,10,>90    C57B1/10           B10.BR    none       9,10,10,10,14,16,20,20    C57B1/10           B10.BR    M7/20.sup.a                                20,27,34,38,>60,>60    C57B1/10           B10.BR    M7/20,day3.sup.b                                11,15,18,>30    C57B1/10           B10.BR    M7/20,day6.sup.c                                19,>30,>30    ______________________________________     .sup.a 5 μg i.p. daily for 10 days     .sup.b 5 μg i.p. daily for 10 days beginning day 3     .sup.c 5 μg i.p. daily for 10 days beginning day 6

The effect of M7/20 on graft rejection was confirmed histologically inseparate groups of C57B/10 recipients of B10.BR heart allograftssacrificed at intervls following transplantation. By three dayspost-transplant control grafts were heavily infiltrated by mononuclearcells. Treatment with M7/20 prevented this graft infiltration. Treatedgrafts at days 6 and 9 had some areas of mononuclear cell infiltration,but markedly less than in control grafts. Both treated and controlgrafts contained considerable myocyte necrosis, evident even at threedays post-transplant. This necrosis appeared unrelated to the rejectionprocess, and may represent ischemic damage sustained duringtransplantation.

The efficacy of M7/20 in reversing established rejection was examined ina small number of C57B1/10 recipients of B10.BR allografts (Table I). Infour animals the onset of treatment was delayed until day 3, by whichtime rejection was ongoing, and continued through day 12. Three graftswere rejected on days 11, 15, and 18, while the fourth was stillfunctioning at 30 days. When treatment was given on days 6-15, one graftwas rejected at 19 days, while two were still functioning at 30 days.

The influence of M7/20 on skin allograft survival was studied in thesame strain combinations used for heart allografts. As seen in theFIGURE, when C57B1/10 skin was placed on B10.AKM recipients, M7/20 at adose of 5μ daily for 10 days significantly prolonged graft survival whencompared with controls (p<0.01). However, none of the skin graftssurvived indefinitely. M7/20 was ineffective in prolonging the survivalof B10.BR skin on C57B1/10 recipients.

RATS Animals and Operative Techniques

Inbred male rats weighing 200-250 g were used throughout(Microbiological Assoc., Walkersville, Md.). Unmodified Lewis rats actedas organ recipients and Lewis-Brown Norway Fl hybrids served as heartdonors. Wistar Furth rats were used as heart donors for specificitystudies.

Heterotopic cardiac grafts were anastomosed to the abdominal greatvessels according to the method of Ono et al. (1969) J. Thorac.Cardiovasc. Surg. 57, 225. The size and ventricular activity wereassessed daily by palpation through the recipient flank. Rejection wastaken as the time of complete cessation of myocardial contractions.

Preparation and Administration of Monoclonal Antibody

A mouse anti-rat IgG, anti-IL-2 receptor monoclonal antibody wasobtained from cultured hybridoma cells (designated ART 18) madeaccording to the method of Kohler and Milstein (1975) Nature 256, 495,as modified by Lemke et al. (1978) Nature 271, 249, and described indetail in Osowa et al. (1983) J. Immunol. 30, 51 (the mice were primedwith phorbol myristate activated rat T-lymphocytes). The antibodyrecognizes the rat 50-kilodalton glycoprotein IL-2 receptor molecule;binds to rat T-lymphoblasts at a rate of 7.5×10⁴ binding sites per cell;does not bind to mature, resting T-lymphocytes; and does not affect thefunctioning of mouse T-lymphocytes not bearing IL-2 receptors.

Antibody (protein concentration 10 mg/ml, 5 mg/ml of pure antibody) wasdiluted in medium and administered to experimental animals intravenouslyat a dose of 25-300 ug of antibody/kg/day for 5 or 10 consecutive days.Alzet osmotic pumps (Model 2 ML1, Alza Corp., Palo Alto, Calif.) wereinserted into the external jugular vein of some recipients to give aconstant infusion of antibody (10.5 ul/hr for 10 days), as is describedin further detail below.

Allograft recipients received anti-IL-2 receptor antibody therapyaccording to arbitrarily chosen doses and durations of treatment.Antibody administered intravenously in doses of 25, 100, or 300ug/kg/day for 10 consecutive days beginning on the day of graftingincreased the mean allograft survival (compared to the untreated 8-daysurvival rate) in a dose dependent fashion to MST±SD=13±1 days, 14±3days, and 21±1 days, respectively. Limiting the period of treatment tothe first five post-transplant days was less effective and resulted in asignificant graft prolongation only when antibody was given at a dose of300 ug/kg/day (14±2 days, p 0.005).

The efficacy of antibody therapy in reversing well established allograftrejection was also tested. Treatment was intitiated at 5 days aftertransplantation, at which time the grafts were grossly enlarged andheavily infiltrated with lymphocytes. Significantly, antibody therapystarted day 5 after transplantation and continued for 5 days at a doseof 300 ug/kg/day improved allograft survival to 18±4 days, a resultcomparable to the effect produced by 10 consecutive injections. Inaddition, the dense cellular infiltrate noted histologically in acuterejection at day 5 had virtually disappeared after the antibody therapy.Intermittent antibody adminstration (5-9 and 15-19 days, with notreatment on days 10-14) extended graft survival even further, to 26-28days, while lower antibody doses were ineffectual in reversing ongoingrejection.

To demonstrate that the results of antibody treatment were not unique toone strain combination, Wistar Furth rat recipients of Lewis cardiacgrafts underwent antibody treatment (300 ug/kg/daily) for 10 daysbeginning the day of transplantation. Allograft survival was prolongedto 16±1 days.

In the next series of experiments, antibody was administered (300ug/kg/day over ten days) intravenously in a constant infusion of 10.5ul/hr using an Alzet osmotic pump. Such treatment was significantly lesseffective than the above-described "pulse" treatment in preventingrejection (graft survival=12-13 days, n=3, p<0.005).

Non-Competition with IL-2

The potentially offsetting effects of exogenously supplied anti-IL-2receptor antibody IL-2 itself were studied in vivo within themicroenvironment of unmodified graft recipients. As shown above, soletherapy with the antibody directed at the rat IL-2 receptor increasescardiac allograft survival to about 3 weeks. In contrast, it has beenpreviously shown that a course of IL-2 accelerates immuneresponsiveness. To test the effects of the two together, the optimaldoses of each (300 ug/kg of antibody and 100 ul of IL-2) were mixed anddelivered in daily intravenous injections for a period of 10 days.Interestingly, this combined treatment produced the same effect as ifantibody had been administered alone (graft survival=20±2 days, n=4),suggesting that an excess of IL-2 does not prevent in vivo binding ofantibody to IL-2 receptor-bearing cells. Moreover, these results suggestthat, in the case of this particular antibody, treatment prolongsengraftment by destroying IL-2 receptor positive cells rather than bypharmacological blocking of the IL-2 receptor.

Effect of Antibody Therapy on T Suppressor Cells

Spleen cells were harvested at day 10 from heart grafted hosts after thedose regimen of anti-IL-2 receptor antibody had been completed, andtransferred intravenously (40-50×10⁶) into normal recipients whichreceived test cardiac allografts 24 hrs later. Such adoptive transferprolonged donor-specific (Lewis-Brown Norway F1 hybrids) but notthird-party (Wistar Furth) test graft survival (15±1 days and 8±1 days,respectively, n=5, p 0.001). Thus, potent antigen specific suppressor,but little alloagressive activity, was demonstrated in animalsmaintaining well-functioning cardiac allografts following antibodytherapy. In other words, the antibody advantageously lysed most (but notall) receptor-bearing T-lymphocytes, but spared the T-suppressor cells,which are important in inhibiting rejection.

Human Dosage and Administration

Dosages of anti-rejection substances will vary, depending on factorssuch as whether or not the substance is lytic, and the condition of thepatient. Generally, lytic monoclonal anti-IL-2 receptor antibodies willbe administered in a series (e.g., two to fifteen more preferably 5 toten intravenous doses, given, e.g., once or twice daily or every two orthree days, or in regular courses interrupted by peroids of cessation oftreatment), begun on the day of the transplant; each dose preferablywill be in the range of about 50-1000 ug/kg. In some instances,treatment initiation can be delayed one or more days following theallograft, since therapy not only can prevent rejection, but can reverseit as well.

OTHER EMBODIMENTS

Other embodiments are within the following claims. For example, thecell-lysing substance can be specific for a growth factor receptor otherthan the IL-2 receptor, provided that the growth factor is presentprimarily on the surfaces of unwanted target cells during aproliferative burst, and is not found on the surfaces of normal cells toan extent which would result in their lysis to an unacceptable extentupon administration of the substance. In the case of the IL-2 receptor,the target cells can be unwanted B-lymphocytes, which, likeT-lymphocytes, bear IL-2 receptors on their surfaces duringproliferative bursts associated, e.g., with acute stages of autoimmunediseases such as systemic lupus erythmatosus. In addition, acute stagesof autoimmune diseases such as multiple sclerosis apparently, likeallograft rejection, involve T-lymphocytes which undergo proliferativebursts associated with the transient appearance of IL-2 receptors, andpatients with such an acute disease can be treated by administering aneffective amount of an IL-2 receptor-specific affinity substance capableof lysing the lymphocytes or interfering with IL-2 binding to them. Thelytic substance, rather than an antibody, can be the growth factoritself (which is highly specific for the growth factor receptor) linkedto a toxin, e.g., ricin or diphtheria toxin.

I claim:
 1. A method of inhibiting T-lymphocyte-induced rejection of anallograft in a mammal, comprising adminstering to said mammal an IL-2receptor specific antibody in an amount effective to inhibit rejectionof said allograft.
 2. The method of claim 1, wherein T-lymphocytes whichexpress IL-2 receptor in response to the antigens of the allograft arecontacted with said IL-2 receptor specific antibody.
 3. The method ofclaims 1 or 2, wherein said IL-2 receptor specific antibody is amonoclonal antibody.
 4. The method of claims 1 or 2, wherein inhibitionof said rejection results from the killing of said T-lymphocytes.
 5. Themethod of claim 4, wherein said killing of said T-lymphocytes occurs bycomplement-fixation.
 6. A method of inhibiting T-lymphocyte inducedrejection of an allograft in a mammal, comprising administering to saidmammal an IL-2 receptor specific antibody in an amount effective toinhibit said rejection, wherein said antibody is administered one ormore times on the day of the allograft or thereafter.
 7. The method ofclaim 6, wherein said antibody is administered one or more times betweenthe day of the allograft and day 19 following the allograft.
 8. Themethod of claim 6, wherein said antibody is administered one or moretimes between days 5 and 19 following the allograft.
 9. The method ofclaim 6, wherein said IL-2 receptor specific antibody is administered ata time when a proliferative burst of T-lymphocytes would occur in theabsence of said antibody.
 10. A method of inhibiting theT-lymphocyte-induced rejection of an allograft in a mammal, comprisingadministering to said mammal, following said allograft, an IL-2 receptorspecific antibody in an amount effective to inhibit rejection of saidallograft.
 11. The method of claim 10, wherein said IL-2 receptorspecific antibody is administered on the day of said allograft.
 12. Themethod of claim 10, wherein said IL-2 receptor specific antibody killssaid T-lymphocytes.
 13. The method of claim 10, wherein said IL-2receptor specific antibody is a complement fixing antibody.
 14. Themethod of claim 10, wherein said IL-2 receptor specific antibody is amonoclonal antibody.
 15. The method of claim 10, wherein said allograftis a skin allograft.
 16. The method of claim 10, wherein said allograftis a transplanted organ.
 17. The method of claim 16, wherein saidtransplanted organ is a heart.